A number of methods have been developed for exponential amplification of nucleic acids. These include the polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (SSR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), and amplification with Qβ replicase (see, e.g., Birkenmeyer et al. (1991) J. Virological Methods 35, 117-126 and Landegren (1993) Trends Genetics 9, 199-202).
Current methods of PCR amplification involve the use of two primers which hybridize to the regions flanking a nucleic acid sequence of interest such that DNA replication initiated at the primers will replicate the nucleic acid sequence of interest. By separating the replicated strands from the template strand with a denaturation step, another round of replication using the same primers can lead to geometric amplification of the nucleic acid sequence of interest. Among other disadvantages, PCR amplification is prone to sequence errors; is limited to amplification of short DNA segments; and cannot proceed continuously, but must be carried out by subjecting the nucleic acid sample to multiple cycles, at shifting temperatures.
Isothermal DNA amplification methods have been described in which such thermal cycling is not required. These methods take advantage of the properties of DNA polymerases such as Φ29 (Phi 29) DNA polymerase, which are highly processive and are able to displace strands of DNA that lie in their path.
One such isothermal method, sometimes termed multiple displacement amplification (MDA), has been used to amplify large molecular weight linear DNA, such as genomic DNA. In one form of this method, two sets of primers are used that are complementary to opposite strands of nucleotide sequences flanking a target sequence. Amplification proceeds by replication initiated at each primer and continuing through the target nucleic acid sequence, with the growing strands encountering and displacing previously replicated strands. In another form of the method, a random set of primers is used to randomly prime a sample of genomic nucleic acid. The primers in the set are collectively, and randomly, complementary to nucleic acid sequences distributed throughout nucleic acid in the sample. Amplification proceeds by replication initiating at each primer and continuing so that the growing strands encounter and displace adjacent replicated strands. In another form of the method concatenated DNA is amplified by strand displacement synthesis with either a random set of primers or primers complementary to linker sequences between the concatenated DNA. Synthesis proceeds from the linkers, through a section of the concatenated DNA to the next linker, and continues beyond, with the growing strands encountering and displacing previously replicated strands.
Rolling circle amplification (RCA) is a multiple displacement amplification method which can be performed on DNA molecules which are circles or which can be circularized and are of a suitable size. In one embodiment, a double-stranded nucleic acid is circularized (e.g. by insertion of a nucleic acid molecule of interest into a linear vector to form a circular molecule) and one strand is nicked, such that one strand is continuous and the other strand is discontinuous. One or more primers are annealed to the continuous strand of the circular vector (a single-stranded circular DNA molecule), and are used to initiate DNA copying. As each primer annealed to the circular template DNA is extended, the growing strand encounters and displaces previously replicated DNA to produce a continuous sequence of tandem copies of the circle. Secondary priming events can subsequently occur, resulting in an exponential amplification. When different primers are used, one or more for each strand, a cascade of strand displacement occurs, resulting in the sort of pinwheel structure shown in FIG. 1. Eventually, the single strands of amplified DNA are converted to double-stranded DNA.
Molecular cloning of DNAs (e.g. having an unknown sequence) is routinely carried out using plasmid, phage, or viral vectors that replicate inside cells. Such methods are sometimes referred to as in vivo cloning methods. Methods of in vivo cloning are described, e.g., in Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. In vivo cloning methods are laborious, requiring steps of electroporating DNA into cells, plating the cells, picking colonies, growing the picked colonies overnight, and preparing template from those cultures. These processes are time consuming, costly and not amenable to high-density formats. Furthermore, certain DNA sequences have proven to be difficult to clone in vivo, because, for example, they are toxic to E. coli or other host cells in which they are to be cloned, or for other reasons that are not well-understood.
Methods of in vitro (cell-free) cloning have been described which obviate some of the problems of in vivo cloning. For example, a method has been described in which individual DNA molecules are cloned in solution by serial dilution and subsequent PCR amplification from tubes containing single molecules (Lukyanov et al. (1996) Nucleic Acid Research 24, 2194-2195. Another method has been described for cloning RNA populations derived from single RNA molecules in an immobilized medium (Chetverina et al. (1993) Nucleic Acids Research 21, 2349-2353). Another method—the “polony” method—has been described for in situ PCR amplification of individual molecules (Mitra et al. (1999) Nucleic Acids Res 27, e34. Among other drawbacks, such methods result in a high mutation rate and stuttering at sequences of low complexity, especially homopolymer tracts.
The inventors describe herein a high fidelity, cell-free method to clone single copies of DNAs (e.g. of unknown sequence), using isothermal rolling circle amplification primed by multiple primers comprising random, partially random or defined sequences.